PB1774: PHARMACOLOGICAL STRATEGIES TO OVERCOME RESISTANCE IN AML CELL LINE MODELS HARBORING ACTIVATING FLT3-ITD AND PTPN11 MUTATIONS
Authors: Maximilian Fleischmann, Nathalie Urban, Ole Hansen, Ulf Schnetzke, Florian Heidel, Andreas Hochhaus, Joerg Mueller, Sebastian Scholl
Journal: HemaSphere (2023)

Topic: 3. Acute myeloid leukemia - Biology & Translational Research Background: Primary or secondary resistance towards various AML-treatment strategies like intensive chemotherapy, tyrosine kinase inhibitors or combination of the BCL-2 inhibitor venetoclax and hypomethylating agents can be mediated by activating mutations of the non-receptor phosphatase PTPN11 (SHP2) or by FLT3-ITD. Both inhibition of FLT3-ITD glycosylation and heat shock protein 90 (HSP90) inhibition reflect promising approaches to overcome these dependencies Aims: We investigated the impact of combined inhibition of glycosylation and HSP90 on cellular signaling pathways and its effectiveness to induce apoptosis by combinational in vitro treatment of Ba/F3 cells in the molecular context of AML-typical and constitutively active FLT3-ITD and/ or PTPN11 mutations. We aimed at distinct pharmacological strategies also considering inhibitors of MEK and STAT5 dependent on cell-type specific activation of signaling pathways. Methods: Murine Ba/F3 leukemia cell lines were stably transfected with either one of two distinct FLT3-ITD variants: FLT3-ITD598/599 (JM domain) vs. FLT3-ITDG613E (TKD1 region) in combination with wildtype PTPN11 or PTPN11-E76K encoding a constitutively active form of this oncogenic phosphatase. Cells were exposed to sequential treatment of the glycosylation inhibitors tunicamycin or 2-deoxy-d-glucose in vitro, either with the HSP90 inhibitor 17-AAG, the STAT5 inhibitor pimozide or the MEK inhibitor trametinib. Protein lysates were consecutively characterized by immunoblotting. Apoptosis and metabolic activity were assessed by Annexin V staining by means of flow cytometry or by Presto Blue assays, respectively. Results: Sequential treatment with tunicamycin and 17-AAG is capable to enhance apoptosis in a synergistic manner in FLT3-ITD/PTPN11 co-transduced Ba/F3 cells, which was significantly more pronounced in FLT3-ITDG613E compared to FLT3-ITD598/599 cells (Table 1). This pharmacological strategy was significantly less effective to induce apoptosis in cells harboring PTPN11-E76K mutations in both FLT3-ITD harboring Ba/F3 cell lines. Based on a pronounced phosphorylation of STAT5 and ERK under tunicamycin treatment in PTPN11-E76K co-mutated cells, sequential incubation with trametinib or pimozide revealed additive apoptosis effects in both FLT3-ITD variants. Pretreatment with tunicamycin was able to enhance sensitivity of both FLT3-ITD cell lines to trametinib or pimozide more than four-fold compared to single agent treatment. (Apoptosis rates of Ba/F3598/599-E76Kvs. Ba/F3G613E-E76K: trametinib 6.43% vs. 5.94%, pimozide 6.28% vs. 6.46%, tunicamycin 15.83% vs. 15.03%, tunicamycin + trametinib 21.75% vs. 23.73%, tunicamycin + pimozide 26.95% vs. 28.15%) - Ba/F3-ITD 589/599 Ba/F3-ITD G613E Relative apoptosis rate GFP-control PTPN11-wildtype PTPN11-E76K GFP-control PTPN11-wildtype PTPN11-E76K DMSO (0.02 v/v %) 1 1 1 1 1 1 Tunicamycin 2.2 2.1 2.1 2.9 3.3 2.2 17-AAG 1.1 1.1 1.1 1.7 1.9 1.3 Tunicamycin + 17AAG 5.6 7.3 3.8 13.5 19.5 6.4 Table 1: Relative apoptosis rates measured by Annexin V of three biological replicates. Summary/Conclusion: The presented Ba/F3 cell line experiments emphasize the potential impact of activating PTPN11 mutations in mediating multi-drug resistance and provide possible approaches of combined targeted therapies to overcome resistance. The combinational use of inhibitors sequentially targeting glycosylation and HSP90 as well as MEK- and STAT5 signaling represents a promising strategy in FLT3-ITD mutated AML cells harboring activating PTPN11 mutations. Keywords: PTPN11, AML, Flt3-ITD, Drug resistance